The present invention relates to tetrapartate prodrugs. In particular, the invention relates to polymer conjugates that provide tetrapartate prodrugs that deliver active agents linked to uptake enhancing moieties effective to provide enhanced efficacy, e.g., as antitumor agents or the like.
Over the years, several methods of administering biologically-effective materials to mammals have been proposed. Many biologically-effective materials, e.g., including medicinal agents and the like, are available as water-soluble salts and can be included in pharmaceutical formulations relatively easily. Problems arise when the desired biologically-effective material is either insoluble in aqueous fluids or is rapidly degraded in vivo. For example, alkaloids are often especially difficult to solubilize.
One way to solubilize biologically-effective material(s) is to include them as part of a soluble prodrug. Thus, prodrugs include chemical derivatives of a biologically-active material, or parent compound which, upon administration, eventually liberate the parent compound in vivo. Prodrugs allow the artisan to modify the onset and/or duration of action of an agent in vivo and can modify the transportation, distribution or solubility of a drug in the body. Furthermore, prodrug formulations often reduce the toxicity and/or otherwise overcome difficulties encountered when administering pharmaceutical preparations.
Typical examples of prodrugs include organic phosphates or esters of alcohols or thioalcohols. See Remington""s Pharmaceutical Sciences, 16th Ed., A. Osol, Ed. (1980), the disclosure of which is incorporated by reference herein.
Prodrugs are, by definition, forms of the parent or active compound. The rate of release of the active drug, typically, but not exclusively, by hydrolysis of the prodrug, is influenced by several factors, but especially by the type of bond joining the active drug to the modifier. Care must be taken to avoid preparing prodrugs which are eliminated through the kidney or reticular endothelial system, etc., before a sufficient amount of the parent compound is released. By incorporating a polymer as part of the prodrug system, one can increase the circulating half-life of the drug. However, in some situations, such as with alkaloids, it has been determined that when only one or two polymers of less than about 10,000 daltons are conjugated thereto, the resulting conjugates are rapidly eliminated in vivo especially if a somewhat hydrolysis-resistant linkage is used. In fact, such conjugates are so rapidly cleared from the body that even if a hydrolysis-prone ester linkage is used, not enough of the parent molecule is regenerated in vivo. This is often not a concern with moieties such as proteins, enzymes and the like, even when hydrolysis-resistant linkages are used. In those cases multiple polymer strands, each having a molecular weight of about 2-5 kDa, are used to further increase the molecular weight and circulating half-life.
One way in which these problems have been addressed is described, for example, by co-owned patent applications Ser. Nos. 09/183,557, filed Oct. 30, 1998, now U.S. Pat. No. 6,180,095 and 08/992,435, filed on Dec. 17, 1997, now abandoned. These teach double prodrugs, i.e., tripartate, that comprise polymer conjugates of various biologically-effective materials, and methods of making these conjugates. The double prodrug linkages are selected to hydrolyze in vivo at a rate which generates sufficient amounts of the xe2x80x9csecondxe2x80x9d and more reactive prodrug compound within a suitable time after administration by, e.g., a 1,4-aryl or 1,6-aryl (e.g, benzyl) elimination reaction, providing improved control of the pharmacokinetics of a number of small molecule drugs, agents and the like. However, further opportunities for particularly selective targeting of diagnostic and/or therapeutic agents to tissues or cells of interest, by means of a rationally designed prodrug conjugate remain.
One particularly desirable target tissue for prodrugs is tumor tissue. It is well known that tumors generally exhibit abnormal vascular permeability characterized by enhanced permeability and retention (xe2x80x9cEPR effectxe2x80x9d). This EPR effect advantageously allows biologically-effective materials, in the form of macromolecules, e.g., protein(s) such as enzymes and/or antibodies and derivatives or fragments thereof, or the like, to readily enter tumor interstitial tissue space (see, for example, the review article by Maeda et al., 2000, J. of Controlled Release, 65:271-284, incorporated by reference herein). Certain other tissues, in addition to tumors, can exhibit this same EPR effect, under conditions of inflammation, and the like.
In brief, and without being bound by any theory or hypothesis as to the working of the EPR effect, it is believed that the EPR effect allows penetration of large molecule or macromolecule substances, including polymer-based delivery systems. This provides a substantially selective delivery of polymer conjugates into tumor tissue space, e.g., tumor interstitial space. Thereafter, however, the same EPR effect is believed to allow the released prodrug(s) and/or any newly released relatively low molecular weight, biologically-effective materials, to rapidly diffuse out of the extracellular tissue space of the targeted tissue. It is believed that if the released active agent fails to be taken up by the surrounding cells at a sufficient rate, they diffuse away from the release site in the ongoing blood or lymphatic flow.
Thus, there continues to be a need to provide additional technologies for forming prodrugs which would benefit from the multiple level prodrug concept and compensate or control for the EPR effect by allowing for more rapid update or transport of the released biologically-effective materials into tumor cells and/or cells of other tissues of interest that exhibit the EPR effect.
Broadly, the invention provides for a tetrapartate prodrug in the form of a compound of Formula I: 
wherein:
L1 is a bifunctional linking moiety.
Broadly, D is a moiety that is a leaving group, or a residue of a compound to be delivered into a cell. More particularly, D is a residue of an active biological material, or H and (y) is a positive integer equal to 1 or greater. Preferably, (y) ranges from 1 to about 5. When (y) is greater than 1, each D moiety is independently selected.
D can be any biologically active material that it is desired to deliver into a target cell or cells of an animal in need of such treatment, including anti-inflammatory agents, detoxifying agents, anticancer agents, and diagnostics for any of these or other conditions.
Preferably, D is an anticancer agent, an anticancer prodrug, a detectable tag, and combinations thereof. Any anticancer agent or suitable tag that can be linked to the tetrapartate prodrug is contemplated. Simply by way of example, these include an anthracycline compound, a topoisomerase I inhibitor, daunorubicin, doxorubicin; p-aminoaniline to name but a few.
When D is a leaving group, D can be, e.g., N-hydroxybenzotriazolyl, halogen, N-hydroxyphthalimidyl, p-nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl and/or thiazolidinyl thione.
Z is covalently linked to [D]y, wherein Z is a moiety that is actively transported into a target cell, a hydrophobic moiety, and combinations thereof. Optionally, Z is monovalent, multivalent, or more preferably, bivalent, wherein (y) is 1 or 2. Z itself optionally includes an amino acid residue, a sugar residue, a fatty acid residue, a peptide residue, a C6-18 alkyl, a substituted aryl, a heteroaryl, xe2x80x94C(xe2x95x90O), xe2x80x94C(xe2x95x90S), and xe2x80x94C(xe2x95x90NR16), where are R16 is as defined below.
When Z includes at least one amino acid residue, the amino acid is, e.g., alanine, valine, leucine, isoleucine, glycine, serine, threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, and/or a combination thereof, to name but a few. When Z includes a peptide, the peptide ranges in size, for instance, from about 2 to about 10 amino acid residues. In one preferred embodiment, the peptide is Gly-Phe-Leu-Gly (SEQ ID NO: 1) or Gly-Phe-Leu.
In addition, Y1 through Y4 are independently O, S, or NR12; and R11 is a mono- or divalent polymer residue.
R1, R4, R9, R10, R12 and R16 are independently hydrogen, C1-6 alkyl, C3-12 branched alkyl, C3-8 cycloalkyl, C1-6 substituted alkyl, C3-8 substituted cycloalkyl, aryl, substituted aryl, aralkyl, C1-6 heteroalkyl, and/or substituted C1-6 heteroalkyl.
R2, R3, R5 and R6 are independently hydrogen, C1-6 alkyl, C1-6 alkoxy, phenoxy, C1-8 heteroalkyl, C1-8 heteroalkoxy, substituted C1-6 alkyl, C3-8 cycloalkyl, C3-8 substituted cycloalkyl, aryl, substituted aryl, aralkyls, halo-, nitro- and cyano-, carboxy-, C1-6 carboxyalkyl and/or substituted C1-6 alkylcarbonyl.
Ar is a moiety which, when included in Formula (I), forms a multi-substituted aromatic hydrocarbon or a multi-substituted heterocyclic group; wherein (m), (r), (s), (t), and (u) are independently zero or one, (p) is zero or a positive integer. In certain preferred embodiments, (p) is 1.
L1 is independently one of the following: 
wherein:
M is X or Q; where X is an electron withdrawing group;
Q is a moiety containing a free electron pair positioned three to six atoms from 
Simply by way of example, Q is one of the following: C2-4 alkyls, cycloalkyls, aryls, and aralkyl groups substituted with a member of the group consisting of NH, O, S, xe2x80x94CH2xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, and/or ortho-substituted phenyls;
X is, for instance any one of O, NR20, 
xe2x80x83S, SO and SO2;
(a) and (n) are independently zero or a positive integer; (b) is zero or one; (g) is a positive integer of 1 or greater;
(q) is three or four;
R7, R8, R14, R15, R17, R18 and R20 are independently selected from the same group as that which defines R1; and Y5 and Y6 are independently O, S, or NR12. It will be appreciated that when (y) is greater than 1, each of the D moieties are the same or different, respectively.
Preferably, (g) ranges from 1 to about 20, or more, but more typically ranges from 1 to about 10.
Optionally, 
comprises an amino acid residue, either naturally occurring or non-naturally occurring.
R11 is a mono- or bivalent polymer, e.g., having a number average molecular weight of from about 2,000 to about 100,000 daltons.
Methods of preparing the tetrapartate prodrugs of the invention are also provided. In one embodiment, the method includes reacting a compound of formula (III): 
with a compound of the formula (IV):
Lxxe2x80x94Zxe2x80x94[D]y;xe2x80x83xe2x80x83(IV)
wherein B is a leaving group for Formula (III) and is defined as above for when D is a leaving group.
Lx is a leaving group for Formula (IV) and is defined as above for when D is a leaving group.
L1, Ar, Z, D, R1-R6, R9-R11, Y1-Y3, and integers are defined as above. The reaction between (III) and (IV) is preferably conducted in the presence of a solvent and a base. The solvent is, for example, chloroform, methylene chloride, toluene, dimethylformamide and/or combinations thereof Dimethylformamide is generally preferred. The base is, for example, dimethylaminopyridine, diisopropylethylamine, pyridine, triethylamine and/or combinations thereof.
Yet another method of preparing a tetrapartate prodrug of the invention is conducted by reacting a compound of formula (V) 
with a biologically active material; wherein
La is a leaving group as defined when D is a leaving group,
L1, Ar, Z, D, R1-R6, R9-R11, Y1-Y3, and integers are defined as above.
The reaction between (V) and the biologically active material is conducted in the presence of a coupling agent, e.g., 1,3-diisopropylcarbodiimide, a dialkyl carbodiimides, 2-halo-1-alkyl-pyridinium halide, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, 1-propanephosphonic acid cyclic anhydride, phenyl dichlorophosphates, and/or combinations thereof. The reaction between (V) and the biologically active material is conducted in the presence of a solvent and a base, e.g., as described above for the previous synthetic method.
Methods of using the inventive tetrapartate prodrugs are also provided, e.g., by treating a disease or disorder in an animal, by administering a pharmaceutically acceptable composition comprising an effective amount of a compound of Formula I, to an animal in need thereof. In particular, a method is provided of delivering a biologically active material D, into a cell in need of treatment therewith, by a process of:
administering a compound of Formula I to an animal wherein the cell is present, and wherein Formula I is hydrolyzed in vivo extracellularly to yield: 
wherein Y* is the remainder of Y2, and is independently selected from the group consisting of HOxe2x80x94, HSxe2x80x94, or HNR12xe2x80x94;
and Formula I-(i) then spontaneously hydrolyzes to 
and CO2,
and Formula I-(iii) Zxe2x80x94[D]y;
wherein Y** is the remainder of Y*, and is independently selected from the group consisting of O, S, or NR12 and
Zxe2x80x94[D]y crosses the membrane of the cell, and is hydrolyzed therein to release D.